Differential localisation and anabolic responsiveness of mTOR complexes in human 1

skeletal muscle in response to feeding and exercise 2

Nathan Hodson1,§, Chris McGlory,2,§, Sara Y. Oikawa2, Stewart Jeromson3, Zhe Song1, Markus A. 3

Rüegg4, D. Lee Hamilton3, Stuart M. Phillips2, Andrew Philp1,* 4

5

1. School of Sport, Exercise and Rehabilitation Sciences, University of Birmingham, UK. 6

2. Department of Kinesiology, McMaster University, Canada 7

3. Faculty of Health Sciences and Sport, University of Stirling, UK. 8

4. Biozentrum, University of Basel, CH. 9

10

* Corresponding author: 11

Andrew Philp, Ph.D. 12

MRC-ARUK Centre for Musculoskeletal Ageing Research 13

School of Sport, Exercise and Rehabilitation Sciences 14

University of Birmingham, Birmingham, UK, B15 2TT 15

Phone: +44 (0) 121 414 8872 Fax: +44 (0) 121 414 4121 Email: a.philp@bham.ac.uk 16

17

§ - Authors contributed equally to this study 18

Running title: mTOR complex distribution in human skeletal muscle 19

Key Words: mTORC1, mTORC2, Raptor, Rictor, Lysosome 20

Articles in PresS. Am J Physiol Cell Physiol (September 27, 2017). doi:10.1152/ajpcell.00176.2017

Copyright © 2017 by the American Physiological Society.

Abbreviations 21

mTOR – Mechanistic target of rapamycin 22

LAMP2 – Lysosomal associated membrane protein 2 23

WGA – Wheat Germ Agglutinin 24

Raptor – Regulatory associated protein of mTOR 25

Rictor – Rapamycin insensitive companion of mTOR 26

GβL - G protein beta subunit-like 27

PRAS40 - Proline-rich AKT1 substrate 1 of 40kDa 28

DEPTOR - DEP domain-containing mTOR-interacting protein 29

mSIN1 - Mammalian stress-activated protein kinase interacting protein 1 30

Protor – Protein observed with Rictor 31

MPS – Muscle protein synthesis 32

MPB – Muscle protein breakdown 33

S6K1 – Ribosomal protein S6 Kinase 1 34

AKT - RAC serine/threonine-protein kinase/ Protein kinase B 35

CASA – Chaperone assisted selective autophagy 36

Rheb – Ras homolog enriched in brain 37

eIF3F - Eukaryotic translation initiation factor 3 subunit F 38

Abstract 39

Mechanistic target of rapamycin (mTOR) resides as two complexes within skeletal muscle. 40

mTOR complex 1 (mTORC1–Raptor positive) regulates skeletal muscle growth, whereas 41

mTORC2 (Rictor positive) regulates insulin sensitivity. To examine the regulation of these 42

complexes in human skeletal muscle, we utilised immunohistochemical analysis to study the 43

localisation of mTOR complexes prior to and following protein-carbohydrate feeding (FED) 44

and resistance exercise plus protein-carbohydrate feeding (EXFED) in a unilateral exercise 45

model. In basal samples, mTOR and the lysosomal marker LAMP2 were highly co-localized 46

and remained so throughout. In the FED and EXFED states, mTOR/LAMP2 complexes were 47

redistributed to the cell periphery (WGA positive staining) (time effect; p=.025), with 39% 48

(FED) and 26% (EXFED) increases in mTOR/WGA association observed 1h post-49

feeding/exercise. mTOR/WGA colocalisation continued to increase in EXFED at 3h (48% 50

above baseline) whereas colocalisation decreased in FED (21% above baseline). A 51

significant effect of condition (p=.05) was noted suggesting mTOR/WGA co-localization was 52

greater during EXFED. This pattern was replicated in Raptor/WGA association, where a 53

significant difference between EXFED and FED was noted at 3h post-exercise/feeding 54

(p=.014). Rictor/WGA colocalization remained unaltered throughout the trial. Alterations in 55

mTORC1 cellular location coincided with elevated S6K1 kinase activity, which rose to a 56

greater extent in EXFED compared to FED at 1h post-exercise/feeding (p<.001), and only 57

remained elevated in EXFED at the 3h time point (p=.037). Collectively these data suggest 58

that mTORC1 redistribution within the cell is a fundamental response to resistance exercise 59

and feeding, whereas mTORC2 is predominantly situated at the sarcolemma and does not 60

alter localisation. 61

62

Introduction 63

Resistance exercise and protein ingestion are potent anabolic stimuli, elevating muscle 64

protein synthesis (MPS) (5, 20) resulting in a positive net protein balance (NPB) (5). Such 65

elevations in MPS are underpinned by the activation of the conserved serine/threonine kinase, 66

mechanistic target of rapamycin (mTOR). This kinase can both augment MPS (17) and offset 67

muscle protein breakdown (MPB) (19). In skeletal muscle, mTOR resides in two distinct 68

complexes distinguishable by the composition of proteins within each. For example, complex 69

1 (mTORC1) contains mTOR, RAPTOR, GβL, PRAS40 and DEPTOR (4), believed to 70

activate protein synthetic machinery (1), whereas complex 2 (mTORC2) is comprised of 71

mTOR, RICTOR, DEPTOR, GβL, Sin1 and Protor, and is implicated in insulin sensitivity 72

and actin cytoskeleton dynamics (4). Due to the critical role mTORC1 plays in regulating 73

protein synthesis, this complex has received the most detailed examination in relation to 74

resistance exercise and protein feeding. Acute resistance exercise, protein ingestion, or 75

combinations of such stimuli are consistently reported to elevate mTORC1 activity (5, 20), 76

with effects maintained for up to 24 hours (6). Furthermore, the acute inhibition of mTORC1 77

with rapamycin administration ablates any effect of anabolic stimuli on MPS (8, 9). 78

As mTORC1 activity seems to be directly implicated in the stimulation of MPS, research has 79

focussed on understanding the mechanism by which mTORC1 is activated. Sancak et al.(21), 80

identified the interaction of mTORC1 with the lysosome to be of particular importance to the 81

activation of the kinase complex in vitro. A similar mechanism has also been reported in 82

rodent skeletal muscle, where eccentric contractions of the tibialis anterior muscle induce 83

mTOR-lysosome colocalisation (13) in parallel to increases in mTORC1 activity (inferred by 84

the phosphorylation of S6K1Thr389). Together these data infer an importance of mTOR-85

lysosome colocalisation in the activation of molecular pathways implicated in protein 86

synthesis. Recently, however, Korolchuk et al. (15) reported that the cellular localisation of 87

these mTOR/lysosomal complexes play a pivotal role in mTOR activation. In support of this 88

hypothesis, we recently reported that a single bout of resistance exercise initiated 89

mTOR/lysosome translocation to the cell periphery, and occurred in parallel to an increase in 90

mTOR activity and interaction between mTOR and proteins involved in translation initiation 91

(23). 92

Whilst the use of immunofluorescence approaches allowed us to study the cellular 93

localisation of mTOR, this approach did not enable us to distinguish between mTOR 94

complexes. Consequently, we were unable to conclude whether the movement of mTOR 95

following anabolic stimuli was mTORC1 or mTORC2 specific. Further, given the parallel 96

group design we employed (23), we were unable to assess whether mTOR translocation was 97

amplified by feeding. Therefore, the aim of the current study was to evaluate whether mTOR 98

translocation following resistance exercise and/or protein-carbohydrate feeding is specific to 99

mTORC1. In addition, we utilised a within-subject design to evaluate whether a synergistic 100

effect of exercise and feeding exists. We hypothesised that exercise plus protein-carbohydrate 101

feeding would elicit a greater mTOR/LAMP2 translocation to the cell periphery compared to 102

feeding alone. Further, we hypothesised this translocation would be specific to mTORC1. 103

Methods 104

Subjects. Eight young, healthy, recreationally active males (age=22.5±3.1y, 105

BMI=24.6±2.2kg/m2, body fat=17.6±4.8%) volunteered to partake in the study. Potential 106

participants were informed about all experimental procedures to be undertaken and any risks 107

involved before written informed consent was obtained. The study was approved by the 108

Hamilton Integrated Research Ethics Board (REB 14-736) and adhered to the ethical 109

standards outlined by the Canadian tri-council policy statement regarding the use of human 110

participants in research as well as the principles according to the Declaration of Helsinki as 111

revised in 2008. 112

Experimental design. Following initial assessment for 1 repetition maximum (1-RM) on leg 113

extension 7 d previously, participants reported to the laboratory at ~7.00am after a 10-h 114

overnight fast. Participants then rested in a semi-supine position on a bed and an initial 115

skeletal muscle biopsy was taken from the vastus lateralis using a modified bergstrom 116

needle. Following this biopsy, participants performed 4 sets of unilateral leg extension 117

(Atlantis, Laval, QC, Canada) at 70% 1RM until volitional failure interspersed by 2 min 118

recovery. Immediately following the cessation of the final set of leg extension all participants 119

consumed a commercially available beverage (Gatorade Recover®, Chicago, IL, USA) that 120

provided 20, 44, and 1g of protein, carbohydrate, and fat respectively. Subsequent bilateral 121

skeletal muscle biopsies were obtained from the vastus lateralis at 1h and 3h after beverage 122

ingestion to examine mTORC1-related signalling and associated localisation. 123

Skeletal muscle immunohistochemistry. Skeletal muscle immunohistochemical preparation 124

and staining was conducted as described previously (23). All samples from each subject were 125

sectioned onto the same slide, in duplicate, to ensure accurate comparisons between time 126

points could be made. 127

Antibodies. The mouse mono-clonal anti-mTOR (#05-1592) antibody was purchased from 128

Merck Chemical Ltd. (Nottingham, UK). The corresponding conjugated secondary antibody 129

to this was Goat anti-mouse IgGγ1 Alexa®594 (#R37121, ThermoFisher, UK). Antibodies 130

targeting LAMP2 (#AP1824d, Abgent, USA), Rictor (CST#53A2, Cell Signalling 131

Technologies, USA) and Raptor (#ab40768, Abcam, Cambridge, UK) were visualised using 132

Goat anti-rabbit IgG(H+L) Alexa®488 secondary antibodies (#A11008, ThermoFisher, UK). 133

Finally, wheat germ agglutinin (WGA-350, #11263, ThermoFisher, UK) was used to identify 134

the sarcolemmal membrane of muscle fibres. 135

Antibody Validation. The specificity of Rictor (CST#53A2) and Raptor (Abcam#ab40768) 136

primary antibodies were tested utilising skeletal muscle samples from the gastrocnemius of 137

muscle-specific knockout (mKO) mice for each protein respectively (3). Wild-type, littermate 138

muscle samples for each mouse model were used as controls. Primary antibodies were also 139

omitted from a subset of samples on slides to examine any background staining from the 140

secondary antibody utilised. The fluorescence intensity of each image was then calculated 141

using ImageJ software (Version 1.51 for Windows). 142

Image capture. Prepared slides were imaged as described previously (23). DAPI UV (340–143

380 nm) filter was used to view WGA-350 (blue) signals and mTOR proteins tagged with 144

Alexa Fluor 594 fluorophores (red) were visualised under the Texas red (540–580 nm) 145

excitation filter. The FITC (465–495nm) excitation filter was used to capture signals of 146

mTOR-complex proteins and LAMP2, which were conjugated with Alexa Fluor 488 147

fluorophores. On average, 8 images were captured per section, and each image contained ~8 148

muscle fibres such that around 120 fibres per time point (per subject) were used for analysis. 149

Image processing and analysis was undertaken on ImagePro Plus 5.1 (Media Cybernetics, 150

MD., USA.) and all factors i.e. exposure time and de-speckling, were kept constant between 151

all images on each individual slide. Image signals generated by WGA were used to estimate 152

cell membrane borders, which were merged with the corresponding target protein images to 153

identify the association between the protein of interest and the plasma membrane. Pearson’s 154

correlation coefficient (Image-Pro software) was used to quantify colocalization with the 155

plasma membrane and mTOR-associated proteins. This process was also completed to 156

quantify the localization of mTOR with complex-associated proteins (Raptor & Rictor) and a 157

marker of the lysosomal membrane (LAMP2). 158

AKT and S6K1 Kinase Activity Assays. At each time point during the experimental trial, a 159

separate piece of muscle tissue was blotted and freed from any visible adipose or connective 160

tissue. The tissue was then frozen in liquid nitrogen and stored at -80ºC. The kinase activity 161

of AKT and S6K1 was determined via [-γ-32P] ATP kinase assays following immuno-162

precipitation of the target protein, as previously described (18). 163

Statistical Analysis. All statistical analysis was conducted on SPSS version 22 for Windows 164

(SPSS Inc., Chicago, IL, USA). Differences in staining intensity between mKO, wild type 165

(WT) and primary omitted (CON) muscle sections were analysed using a one-way analysis of 166

variance (ANOVA). Differences in kinase activity, fluorescence intensity and staining 167

colocalisation were analysed using a two-factor mixed-model ANOVA with two within 168

subject factors (time; three levels – PRE.vs.1h.vs.3h and condition; two levels – 169

FEDvs.EXFED), with Bonferonni correction for multiple comparisons. Pairwise comparisons 170

were conducted when a significant main/interaction effect was found. Significance for all 171

variables analysed was set at p≤.05. Data are presented as means±SEM unless otherwise 172

stated. 173

Results 174

Rictor and Raptor antibodies are specific to their target proteins. Rictor protein staining 175

intensity in Rictor mKO tissue was significantly lower than that in littermate WT controls 176

(p<.001, Fig.1B). Furthermore, the staining intensity in this tissue was comparable to when 177

the primary antibody was omitted in both mKO and WT tissue (p>.999, Fig.1B). Raptor 178

protein staining intensity in Raptor mKO tissue was also significantly lower than that noted in 179

littermate WT controls (p<.001, Fig.1C), with this staining intensity again similar to when the 180

primary antibody was omitted in either tissue (p>.999, Fig.1C). Therefore, we take this as 181

evidence that the Rictor (CST#53A2) and Raptor (Abcam#ab40768) antibodies are specific 182

to their target protein. 183

S6K1 and AKT kinase activity. A significant condition by time effect was observed for S6K1 184

activity (p<.001). S6K1 activity rose above baseline in both conditions at 1h post-185

exercise/feeding (FED-p=.015, EXFED-p<.001), and kinase activity at this time point was 186

165% greater in the EXFED condition (p<.001, Fig.1D). At 3h post-exercise/feeding, kinase 187

activity only remained above baseline values in the EXFED condition (52.8% greater than 188

baseline, p=.037, Fig.1D). A significant main effect for time was noted for AKT kinase 189

activity (p=.023, Fig.1E). Pairwise comparisons displayed a trend toward an increase in AKT 190

kinase activity 1h post-intervention, when conditions were combined, compared to 3h post-191

intervention (p=.073). 192

Lysosomal content and colocalisation with mTOR. LAMP2 fluorescence intensity was 193

unchanged from baseline in either condition, however a significantly greater intensity was 194

noted in the EXFED condition, compared to FED, at 3h post-exercise/feeding (p=0.41, 195

Fig.2B). A significant condition u time effect was observed for mTOR-Lamp2 colocalisation 196

(p=.004). Consistent with our previous work (23), mTOR and LAMP2 were highly localised 197

in basal skeletal muscle (Fig.2C). The colocalisation of these two proteins did not change 198

from baseline in either condition over the 3h post-exercise/feeding period. However, at the 3h 199

time point, the colocalisation of the proteins was greater in the FED condition compared to 200

the EXFED condition (0.51(FED)vs.0.47(EXFED), p=.011, Fig. 2C). 201

mTOR/lysosome translocation to the cell membrane. Significant main effects of condition 202

(p=.05) and time (p=.025) were observed for mTOR colocalisation with the cell membrane 203

(WGA positive staining). The significant main effect of condition suggests that, when all 204

time points are combined, mTOR-WGA was greater in the EXFED condition compared to 205

the FED condition. Subsequent pairwise comparisons also display that when both conditions 206

were combined, mTOR colocalisation with the cell membrane was greater at 3h post-207

exercise/feeding compared to baseline values (p=.008, Fig.2C). Further comparisons also 208

displayed a trend toward a difference between mTOR-WGA colocalisation between 209

conditions at the 3h time point (0.16(FED) vs. 0.19(EXFED), p=.085). This pattern of 210

colocalisation was mirrored when analysing LAMP2-WGA colocalisation (main effect of 211

time, p=.031, data not shown.), reiterating the constant colocalisation of mTOR and the 212

lysosome. 213

Rictor colocalisation with mTOR and WGA. Significant main effects of group (p=.046) and 214

time (p=.035) were noted for Rictor colocalisation with mTOR proteins (Fig.3B). Overall, 215

there was a greater colocalisation of these two proteins in the EXFED condition compared to 216

the FED condition. Following pairwise comparisons, there was no difference in the 217

colocalisation between Rictor and mTOR between any time points (p>.05, Fig.3B). 218

Furthermore, Rictor colocalisation with WGA did not change from baseline at any time point 219

in either condition (Fig.3C), suggesting post exercise translocation is specific to mTORC1. 220

Raptor colocalisation with mTOR and WGA. The colocalisation of Raptor and mTOR 221

proteins did not change in either group, at any time point, suggesting any alterations in sub-222

cellular location of either protein occurred concurrently (Fig.4B). A significant condition x 223

time effect was observed for Raptor colocalisation with WGA (p=.029). Here, Raptor 224

colocalisation with WGA rose to a similar extent to the previously reported increase in 225

mTOR-WGA colocalisation at 1h post-exercise/feeding in both conditions. At the 3h time 226

point, Raptor-WGA colocalisation in the FED group dropped below baseline and 1h post-227

ex/feeding levels (p=.007, Fig.4C), and colocalisation at this time point was greater in the 228

EXFED condition (0.12(FED) vs. 0.17(EXFED), p=.014, Fig.4C). 229

Discussion 230

Utilising a within-subject design, we report that a combination of unilateral resistance 231

exercise and protein-carbohydrate feeding elicits a greater mTOR translocation toward the 232

cell membrane than feeding alone. This observation is consistent with previous findings from 233

our laboratory in which we reported that mTOR associates with the lysosome in basal skeletal 234

muscle, with mTOR/lysosomal complexes translocating to the cell periphery following 235

mTOR activation (23). Utilising immunofluorescent approaches to distinguish between 236

mTORC1 and mTORC2, the present study extends this observation, suggesting that 237

mTORC1 seems to be the predominant mTOR complex translocating in human skeletal 238

muscle following anabolic stimuli, with mTORC2 in constant association with the cell 239

membrane. 240

In addition to mTORC1 translocation to the cell periphery, we report a greater colocalisation 241

of mTOR and LAMP2 in the FED condition, compared to the EXFED condition, at the 3h 242

time point. This finding was unexpected and contrasted our previous research using a parallel 243

group design (23). The greater association of mTORC1 with lysosomes in the FED condition 244

would infer greater mTORC1 activity in this leg (13, 21); however, this was not apparent in 245

our S6K1 kinase activity data. A possible explanation for this difference is the increased 246

lysosomal content (LAMP2 fluorescence intensity) noted in the EXFED condition at this time 247

point (Fig.2B). It is possible that the acute resistance exercise bout may have elicited an 248

increase in chaperone assisted selective autophagy as a stress response to the strenuous 249

exercise, as previously reported (24). This may have increased the free-lysosomal pool (24) 250

and altered the ratio of mTOR-LAMP2 association. As this is only a proxy measure of 251

lysosomal content, further research directed towards lysosomal biogenesis in response to 252

physiological stimuli would be needed to address this mechanism. 253

Previous research from our laboratory has shown an elevation in mTOR association with the 254

cell membrane in response to resistance exercise, in both the fed and fasted state (23). This 255

association coincided with an increase in S6K1 kinase activity, suggesting that mTOR 256

trafficking is associated with an increase in intrinsic mTOR activity. Consistent with this 257

hypothesis, here we report that mTORC1-cell membrane association increased 1h post-258

intervention, in both FED and EXFED conditions, and the increment was similar to that noted 259

in our previous work (23). However, in contrast to our previous results, mTOR-WGA 260

colocalisation in the FED condition returned close to baseline values at 3h and colocalisation 261

in the EXFED condition displayed a continued elevation. In addition to the main effects of 262

time (p=.025) and condition (p=.05) apparent here, a trend toward greater colocalisation in 263

the EXFED condition (p=.085) was noted at the 3h time point. This greater colocalisation is 264

suggestive of retention of mTOR at the cell periphery when resistance exercise is followed 265

with protein/carbohydrate ingestion, inferring a synergistic effect of resistance exercise and 266

protein-carbohydrate feeding, an observation previously reported for MPS (5). 267

The mTORC1 and mTORC2 protein complexes are involved in varying metabolic signalling 268

processes in skeletal muscle, and as such are suggested to reside in distinct cellular locations 269

(4). As mTOR-lysosome translocation has been previously associated with mTORC1 270

activation in response to amino-acids in vitro (15), we sought to determine whether mTORC1 271

is the principal mTOR complex translocating in human skeletal muscle as we have previously 272

reported (23).The colocalisation of Raptor with WGA increased at 1h post-intervention in 273

both conditions, and to a similar extent to that noted in mTOR-WGA colocalisation, 274

suggesting that mTORC1 is a spatially regulated mTOR complex in human skeletal muscle. 275

Further to this notion, a disparity between conditions became apparent at the 3h time point, 276

with Raptor-WGA colocalisation enhanced in the EXFED condition (p=.014). This is in 277

agreement with the data regarding mTOR-WGA colocalisation where a trend toward EXFED 278

eliciting greater membrane colocalisation compared to FED is reported. Raptor colocalisation 279

with mTOR itself was not altered at any time point, or between conditions, however we did 280

observe a reduction in raptor association with WGA at 3h in both FED and EXFED. We are 281

currently unable to explain this result, however, it could be due to an increase in free Raptor 282

content (14) or increased Raptor degradation (12), both potential mechanisms proposed to 283

regulate mTORC1 activity. In contrast, co-staining of Rictor with WGA, suggested that 284

mTORC2 localises with the cell membrane in basal tissue, with this colocalisation unaffected 285

by resistance exercise or protein/carbohydrate ingestion. This finding has also been replicated 286

using in vitro models, where a large proportion of mTORC2 activity was noted at the plasma 287

membrane of HEK293 cells (10). 288

Our data are congruent with both in vitro (15) and in vivo (23) studies suggesting that 289

mTORC1 cellular colocalisation is linked to mTORC1 activity. Whilst this observation is in 290

contrast to previous in vitro studies (21, 25), where mTOR translocation to the lysosome is 291

deemed essential, we believe the increase in autophagy/MPB in post-absorptive skeletal 292

muscle prevents the disassociation of mTOR and the lysosome noted in previous in vitro 293

studies, where a complete amino acid withdrawal protocol is utilised. Further, many 294

physiological mechanisms occur at the cell periphery suggesting the redistribution of 295

mTORC1/lysosomal complexes to the cell periphery is physiologically relevant. mTORC1 is 296

known to stimulate MPS which, through the use of the SUnSET technique (22) and 297

immunohistochemical staining methods, is purported to occur primarily in peripheral regions 298

of muscle fibres (11). Consistent with this, we previously identified mTOR to interact with 299

Rheb, eIF3F and the microvasculature at the cell periphery following resistance exercise in 300

the fed state (23). Collectively this data therefore suggests that both upstream regulators and 301

downstream substrates of mTORC1 are membrane-associated in skeletal muscle (11). 302

Further, given we observed that mTORC1 association with the cell periphery was prolonged 303

with feeding [a scenario of heightened MPS], we propose that maintaining mTORC1 at the 304

cell periphery may provide an mechanistic explanation as to why exercise in the fed state 305

results in prolonged increases in MPS in human skeletal muscle compared to exercise or 306

feeding in isolation (6). 307

In summary, our data show that mTOR-lysosome translocation in response to resistance 308

exercise and feeding is driven primarily by mTORC1, and occurs in parallel to increases in 309

S6K1 activity. Further, we report that resistance exercise combined with protein-carbohydrate 310

feeding sustains this response, compared to feeding alone, suggesting a synergistic effect of 311

these two stimuli. Collectively, these data add further support to the importance of spatial 312

regulation of mTORC1 in response to anabolic stimulation. Further research should now 313

examine the relevance of mTORC1 colocalisation in clinical scenarios, i.e ageing (7) or 314

obesity (2). Finally, the tools described herein to study mTORC2 localisation could be used 315

to examine the regulation of skeletal muscle glucose uptake and insulin sensitivity, factors 316

thought to be under the direct control of mTORC2 (16). 317

318

Grants: This study was funded in part by a BBSRC New Investigator Award BB/L023547/1 319

to AP and a University of Birmingham ‘Exercise as Medicine’ doctoral training studentship 320

to NH. Work in the laboratory of MAR was supported by the Swiss National Science 321

Foundation and the University of Basel. Work in the laboratory of DLH was funded by a 322

Society for Endocrinology Early Career Grant. Work in the laboratory of SMP was funded by 323

the National Science and Engineering Research Council of Canada and the Canada Research 324

Chairs Program. 325

326

Disclosures: The author(s) declare no competing financial interests. 327

328

Author Contributions: NH and AP conceived the study. NH analysed data presented in 329

Figures 1-4. CM, SO and SMP conducted human experiments reported in Figures 2-4. SJ, ZS 330

and DLH performed experiments and analysis presented in Fig 1. MAR generated the Raptor 331

and Rictor mKO mice used in figure 1. NH and AP interpreted results of experiments. NH 332

conducted statistical analysis and prepared figures. NH and AP drafted the manuscript, with 333

all authors approving the final version of the manuscript. 334

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1. Baar K, and Esser K. Phosphorylation of p70(S6k) correlates with increased skeletal muscle 336 mass following resistance exercise. The American journal of physiology 276: C120-127, 1999. 337 2. Beals JW, Sukiennik RA, Nallabelli J, Emmons RS, van Vliet S, Young JR, Ulanov AV, Li Z, 338 Paluska SA, De Lisio M, and Burd NA. Anabolic sensitivity of postprandial muscle protein synthesis 339 to the ingestion of a protein-dense food is reduced in overweight and obese young adults. The 340 American journal of clinical nutrition 104: 1014-1022, 2016. 341 3. Bentzinger CF, Romanino K, Cloetta D, Lin S, Mascarenhas JB, Oliveri F, Xia J, Casanova E, 342 Costa CF, Brink M, Zorzato F, Hall MN, and Ruegg MA. Skeletal muscle-specific ablation of raptor, 343 but not of rictor, causes metabolic changes and results in muscle dystrophy. Cell metabolism 8: 411-344 424, 2008. 345 4. Betz C, and Hall MN. Where is mTOR and what is it doing there? The Journal of cell biology 346 203: 563-574, 2013. 347 5. Biolo G, Tipton KD, Klein S, and Wolfe RR. An abundant supply of amino acids enhances the 348 metabolic effect of exercise on muscle protein. The American journal of physiology 273: E122-129, 349 1997. 350 6. Burd NA, West DW, Moore DR, Atherton PJ, Staples AW, Prior T, Tang JE, Rennie MJ, Baker 351 SK, and Phillips SM. Enhanced amino acid sensitivity of myofibrillar protein synthesis persists for up 352 to 24 h after resistance exercise in young men. The Journal of nutrition 141: 568-573, 2011. 353 7. Cuthbertson D, Smith K, Babraj J, Leese G, Waddell T, Atherton P, Wackerhage H, Taylor 354 PM, and Rennie MJ. Anabolic signaling deficits underlie amino acid resistance of wasting, aging 355 muscle. FASEB journal : official publication of the Federation of American Societies for Experimental 356 Biology 19: 422-424, 2005. 357 8. Dickinson JM, Fry CS, Drummond MJ, Gundermann DM, Walker DK, Glynn EL, Timmerman 358 KL, Dhanani S, Volpi E, and Rasmussen BB. Mammalian target of rapamycin complex 1 activation is 359 required for the stimulation of human skeletal muscle protein synthesis by essential amino acids. 360 The Journal of nutrition 141: 856-862, 2011. 361 9. Drummond MJ, Fry CS, Glynn EL, Dreyer HC, Dhanani S, Timmerman KL, Volpi E, and 362 Rasmussen BB. Rapamycin administration in humans blocks the contraction-induced increase in 363 skeletal muscle protein synthesis. The Journal of physiology 587: 1535-1546, 2009. 364

10. Ebner M, Sinkovics B, Szczygiel M, Ribeiro DW, and Yudushkin I. Localization of mTORC2 365 activity inside cells. The Journal of cell biology 216: 343-353, 2017. 366 11. Goodman CA, Mabrey DM, Frey JW, Miu MH, Schmidt EK, Pierre P, and Hornberger TA. 367 Novel insights into the regulation of skeletal muscle protein synthesis as revealed by a new 368 nonradioactive in vivo technique. FASEB journal : official publication of the Federation of American 369 Societies for Experimental Biology 25: 1028-1039, 2011. 370 12. Han K, Xu X, Xu Z, Chen G, Zeng Y, Zhang Z, Cao B, Kong Y, Tang X, and Mao X. SC06, a novel 371 small molecule compound, displays preclinical activity against multiple myeloma by disrupting the 372 mTOR signaling pathway. Sci Rep 5: 12809, 2015. 373 13. Jacobs BL, You JS, Frey JW, Goodman CA, Gundermann DM, and Hornberger TA. Eccentric 374 contractions increase the phosphorylation of tuberous sclerosis complex-2 (TSC2) and alter the 375 targeting of TSC2 and the mechanistic target of rapamycin to the lysosome. The Journal of 376 physiology 591: 4611-4620, 2013. 377 14. Kim K, and Pajvani UB. "Free" Raptor - a novel regulator of metabolism. Cell cycle 378 (Georgetown, Tex) 15: 1174-1175, 2016. 379 15. Korolchuk VI, Saiki S, Lichtenberg M, Siddiqi FH, Roberts EA, Imarisio S, Jahreiss L, Sarkar S, 380 Futter M, Menzies FM, O'Kane CJ, Deretic V, and Rubinsztein DC. Lysosomal positioning coordinates 381 cellular nutrient responses. Nature cell biology 13: 453-460, 2011. 382 16. Lamming DW, Ye L, Katajisto P, Goncalves MD, Saitoh M, Stevens DM, Davis JG, Salmon 383 AB, Richardson A, Ahima RS, Guertin DA, Sabatini DM, and Baur JA. Rapamycin-induced insulin 384 resistance is mediated by mTORC2 loss and uncoupled from longevity. Science (New York, NY) 335: 385 1638-1643, 2012. 386 17. Marcotte GR, West DW, and Baar K. The molecular basis for load-induced skeletal muscle 387 hypertrophy. Calcified tissue international 96: 196-210, 2015. 388 18. McGlory C, White A, Treins C, Drust B, Close GL, Maclaren DP, Campbell IT, Philp A, Schenk 389 S, Morton JP, and Hamilton DL. Application of the [gamma-32P] ATP kinase assay to study anabolic 390 signaling in human skeletal muscle. Journal of applied physiology (Bethesda, Md : 1985) 116: 504-391 513, 2014. 392 19. Meijer AJ, Lorin S, Blommaart EF, and Codogno P. Regulation of autophagy by amino acids 393 and MTOR-dependent signal transduction. Amino acids 47: 2037-2063, 2015. 394 20. Phillips SM, Tipton KD, Aarsland A, Wolf SE, and Wolfe RR. Mixed muscle protein synthesis 395 and breakdown after resistance exercise in humans. The American journal of physiology 273: E99-396 107, 1997. 397 21. Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, and Sabatini DM. Ragulator-Rag 398 complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. 399 Cell 141: 290-303, 2010. 400 22. Schmidt EK, Clavarino G, Ceppi M, and Pierre P. SUnSET, a nonradioactive method to 401 monitor protein synthesis. Nature methods 6: 275-277, 2009. 402 23. Song Z, Moore DR, Hodson N, Ward C, Dent JR, O’Leary MF, Shaw AM, Hamilton DL, Sarkar 403 S, Gangloff Y-G, Hornberger TA, Spriet LL, Heigenhauser GJ, and Philp A. Resistance exercise 404 initiates mechanistic target of rapamycin (mTOR) translocation and protein complex co-localisation 405 in human skeletal muscle. Scientific Reports 7: 5028, 2017. 406 24. Ulbricht A, Gehlert S, Leciejewski B, Schiffer T, Bloch W, and Hohfeld J. Induction and 407 adaptation of chaperone-assisted selective autophagy CASA in response to resistance exercise in 408 human skeletal muscle. Autophagy 11: 538-546, 2015. 409 25. Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, and Sabatini DM. mTORC1 senses 410 lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. 411 Science (New York, NY) 334: 678-683, 2011. 412

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Figure headings

Figure 1. Rictor and Raptor antibody validation and S6K1 and AKT kinase activity.

Immuno-fluorescent staining of each protein was performed in mKO and littermate WT

samples, in addition to staining of each sample with primary antibodies omitted (CON).

Rictor/Raptor is displayed in green and WGA (cell membrane) is stained in blue.

Representative images of staining in each condition are displayed (A) alongside the

corresponding quantifications for Rictor (B) and Raptor (C). Scale bars are 50µm. Data

presented as mean±SEM. *Significantly different WT (p<.001). S6K1 (D) and AKT (E)

kinase activity following unilateral resistance exercise and/or protein-carbohydrate feeding.

Black bars denote FED condition and open bars denote EXFED condition. Data presented as

Mean±SEM. *Significantly different to baseline (p<.05), ¥significant difference between

conditions at this time point (p<.001).

Figure 2. The effect of resistance exercise and/or protein carbohydrate feeding on

mTOR-LAMP2 and mTOR-WGA colocalisation. Representative images of mTOR-

LAMP2 and mTOR-WGA co-localisation at rest, and following resistance exercise and/or

protein-carbohydrate feeding (A). Orange/yellow regions denote areas of mTOR localisation

with the marker of the lysosome in images on the top row. mTOR-positive staining is shown

in red, LAMP2-positive in green and WGA-positive in blue. Quantification of LAMP2

fluorescence intensity (B), mTOR-LAMP2 colocalisation (C) and mTOR-WGA (D) co-

localisation at each time point. Scale bars are 50µm. Data presented as Mean±SEM.

¥Significant difference between conditions at this time point (p<.05), #significantly difference

compared to baseline when conditions combined (p=.008).

Figure 3. The effect of resistance exercise and/or protein carbohydrate feeding on

Rictor-mTOR and Rictor-WGA colocalisation. Representative images of Rictor-mTOR

and Rictor-WGA co-localisation at rest, and following resistance exercise and/or protein-

carbohydrate feeding (A). Orange/yellow regions denote areas of Rictor localisation with

mTOR on top row. mTOR-positive staining is shown in red, Rictor-positive in green and

WGA-positive in blue Quantification of Rictor-mTOR (B) and Rictor-WGA (C) co-

localisation at each time point. Scale bars are 50µm. Data presented as Mean±SEM.

Figure 4. The effect of resistance exercise and/or protein carbohydrate feeding on

Raptor-mTOR and Raptor-WGA colocalisation. Representative images of Raptor-mTOR

and Raptor-WGA co-localisation at rest, and following resistance exercise and/or protein-

carbohydrate feeding (A). Orange/yellow regions denote areas of Raptor localisation with

mTOR. mTOR-positive staining is shown in red, Raptor-positive in green and WGA-positive

in blue Quantification of Raptor-mTOR (B) and Raptor-WGA (C) co-localisation at each

time point. Scale bar is 50µm. Data presented as Mean±SEM. ¥Significant difference between

conditions at this time point (p=014), #significantly difference compared to baseline when

conditions combined (p=.007).